The coat protein complex II (COPII) mediates the sorting and export of cargo proteins from the endoplasmic reticulum (ER). COPII serves as a sorting device, selecting cargo proteins for incorporation into budded vesicles. COPII also functions as a mechanical device to form and release vesicles. In cells, COPII is organized at discrete sites on ER membranes; ER exit sites (ERES). Accessory COPII-binding proteins control the organization and activity of COPII at these sits. We combine highly defined in vitro experimental systems that reconstitute COPII activities on synthetic membranes and ER membranes with in vivo studies to provide a mechanistic view of the COPII-mediated ER export event. In aim 1, we will examine the physical role of COPII proteins in vesicle release. We will examine the hypothesis that Sar1 functions in a GTPase regulated manner to drive membrane fission. We will use highly defined vitro fission assays and measurements on ER membranes to determine the role of Sar1 and the COPII cage in this process. In aim 2 we will examine the hypothesis that lipid signals are recognized by coat binding proteins to regulate COPII activities at ERES. We will focus on a candidate protein, the Sec31 and Sec23 interacting protein p125A. P125A is a PA specific phospholipase A1 homolog that regulates COPII nucleation and traffic from ERES. We will perform structure- function analysis using in vitro biochemical studies and dynamic cellular studies to define the role of p125A in decoding lipid signals and regulating COPII activities. In aim 3 we will examine a novel mechanism for the activation of lipid remodeling enzymes such as the phosphatidylinositol (PI) 4- kinase II? at ERES. Specifically we hypothesize that the physical perturbation of membranes by Sar1 (studied in aim 1) is utilized to couple membrane curvature with enzyme activity. We will further define the selective contributions of PI 4- kinases II? and III? in regulating COPII at ERES in response to different cargo loads. Importantly, a large number of human diseases are derived from mistakes in protein sorting at the ER. Developing mechanistic understanding of the molecular basis for COPII functions in ER export should enable the development of future effective therapeutics. PUBLIC HEALTH RELEVANCE: The endoplasmic reticulum (ER) is a sub cellular compartment that functions as an assembly factory for proteins that are expressed on the cell surface or secreted from the cell. About a third of the proteins encoded by the human genome are assembled in the ER. Many seemingly unrelated human diseases are derived from mistakes in the shipment of assembled proteins from the ER. In our studies we explore the role of proteins that mediate this critical selection and shipment step. Specific proteins assemble the vesicles in which proteins to be shipped are selectively packaged. We reconstitute the vesicle formation process with pure proteins in cell- free test tube settings. Experiments in this highly defined system provide insights that are than utilized for analysis in cells. Developing mechanistic understanding of the molecular basis for protein selection and export from the ER should enable the development of future effective therapeutics.